Many opportunistic pathogens can colonize human tissue or artificial implant devices leading to biofilm associated bacterial infections. Bacteria growing in biofilms often have increased resistances to antimicrobials and to host defensive processes, when compared to the same strain growing in planktonic culture. The mechanisms for these enhanced resistances are not well understood. Since most antibiotics fully penetrate microbial biofilms, persistence of biofilm infections likely involves physiological differences of the biofilm-associated cells compared to their more antibiotic-sensitive and host-susceptible planktonic counterparts. Progress has been made on the transcriptomic and proteomic differences between biofilm associated and planktonic cells. However, since bacteria growing in biofilms do not experience homogenous environmental conditions, the cells are not physiologically identical throughout the biofilm. Therefore, to gain an understanding of the factors that allow biofilm persistence in infectious diseases, it is essential to identify and characterize novel factors that are expressed at spatially localized sites within biofilms. To initiate these studies, we have designed experiments to characterize the physiological heterogeneities of subsets of bacterial cells within biofilms. We will combine laser capture microdissection microscopy and molecular approaches to identify and quantify the expression of genes at localized sites within biofilms. In particular we will: (i) develop analytical strategies necessary to quantify gene expression at cellular level within biofilms, (ii) characterize spatial transcription gradients of genes known to be important during Pseudomonas aeruginosa biofilm growth, virulence, and antibiotic resistance, (iii) develop the protocols necessary to perform global transcriptomic studies on spatially localized cells within microbial bioftlms. Our long term goals are to identify targets for anti-biofilm therapies, and to understand how localized expression of these targets affect bacterial virulence, antibiotic tolerance, and persistence of bacteria in biofilms. [unreadable] [unreadable] [unreadable]